This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Microsporidia are obligately intracellular, single-celled fungal parasites that infect invertebrate and vertebrate hosts. Infections due to microsporidia occur worldwide in both immune-deficient and immune-competent humans of all age groups and are associated with diarrhea and systemic disease. Molecular-based PCR methods have improved sensitivity and specificity for diagnosing microsporidiosis but require costly equipment (eg. thermocycler) and take hours to perform. In this study, an accelerated LAMP method was applied to improve the time efficiency for molecular detection of the two most prevalent microsporidia species infecting humans, Enterocytozoon bieneusi and Encephalitozoon intestinalis. Primer Explorer LAMP software (http://primerexplorer.jp/e/v3_manual/index.html) was used to select primers from the ribosomal RNA gene targets of Ent. bieneusi (Genbank L07123) and Enc. intestinalis (Genbank L19567). Specimens tested included Ent. bieneusi and Enc. intestinalis spores obtained from rhesus macaque bile and tissue culture supernatants that were suspended in tissue culture medium or spiked into stool. Extracted DNA samples were reacted with 0.8 uM FIP and BIP primers, 0.2 uM F3 and B3 primers, 0.4 uM LF and LB loop primers, 1.5 M betaine, 16 units Bst polymerase, and 0.5 M dNTPs in Bst buffer at a final volume of 50 ul for 1 hour at 63? C followed by 10 min enzyme deactivation at 80? C for the LAMP or via nested PCR using rDNA primers. Sensitivity of nested PCR was 1 ?10 spores per reaction volume (or 10 ?100 spores / ml feces). LAMP was a log less sensitive at 10 ?100 spores per reaction volume or 100 ?1000 spores per ml feces. Attempts are in progress to improve sensitivity of LAMP using FTA Whatman filter papers for fixation of microsporidia DNA prior to extraction and amplification in attempt to remove inhibitors. These results support further development of LAMP due to its lower cost, shorter time, higher specificity, and no requirement for special equipment when compared to nested PCR, but sensitivity of detection with LAMP needs to be improved.
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